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Aop: 260

AOP Title

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CYP2E1 activation and formation of protein adducts leading to neurodegeneration

Short name:

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CYP2E1 activation and formation of protein adducts leading to neurodegeneration

Graphical Representation

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Authors

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Point of Contact

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Jelle Broeders   (email point of contact)

Contributors

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  • Jelle Broeders
  • Marvin Martens

Status

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Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite


This AOP was last modified on April 09, 2018 11:53

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Revision dates for related pages

Page Revision Date/Time
CYP2E1 Activation April 09, 2018 11:02
Protein Adduct Formation March 26, 2018 09:45
Oxidative Stress in Brain April 04, 2018 14:24
Lipid Peroxidation April 04, 2018 14:33
Unfolded Protein Response April 04, 2018 14:38
General Apoptosis April 04, 2018 14:51
Neurodegeneration April 04, 2018 14:53
CYP2E1 Activation leads to Oxidative Stress in Brain April 05, 2018 03:40
Oxidative Stress in Brain leads to Lipid Peroxidation April 05, 2018 04:06
Lipid Peroxidation leads to Protein Adduct Formation April 05, 2018 04:18
Protein Adduct Formation leads to Unfolded Prortein Response April 05, 2018 04:25
Oxidative Stress in Brain leads to Unfolded Prortein Response April 05, 2018 04:37
Lipid Peroxidation leads to General Apoptosis April 05, 2018 04:48
Unfolded Prortein Response leads to General Apoptosis April 05, 2018 04:51
General Apoptosis leads to Neurodegeneration April 05, 2018 04:53
Acetaminophen November 29, 2016 18:42
Enflurane April 05, 2018 06:31
Halothane April 05, 2018 06:32
Isoflurane April 05, 2018 06:32
Methoxyflurane April 05, 2018 06:32
Sevoflurane April 05, 2018 06:33
Chemical:584015 (1-~13~C)Aniline April 05, 2018 06:33
Chlorzoxazone April 05, 2018 06:34
Titanium oxide (TiO) April 05, 2018 06:35
Isoniazid April 05, 2018 06:36
Ethanol April 05, 2018 06:38

Abstract

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The AOP has two different MIEs: protein adduct formation (MIEa) and CYP2E1 activation (MIEb). Protein adduct formation is the interaction between a chemical, or reactive metabolite, and a protein at molecular level. During this interaction a covalent bond is formed which occurs due to the reaction between an electrophilic chemical and the nucleophilic part of a protein. When a chemical forms a covalent bond with a protein the protein is damaged and can loses its function. Acetaldehyde, the metabolite of ethanol, is also one of these chemicals known to form protein adducts. This is why protein adduct formation is added in this AOP based on ethanol. CYP2E1 is one of the enzymes responsible for the metabolism of ethanol, and because of this metabolic activity the MIE in added in this AOP. CYP2E1 participates in the metabolism of endogenous, small and hydrophobic compounds using a oxidation reaction. CYP2E1 is mainly expressed in rat liver cells, but can also be found in rat brain cells. Furthermore, in the human brain CYP2E1 expression is mainly found in the amygdala and prefrontal cortex. At higher concentrations of ethanol the expression of CYP2E1 increases, as well as the activity of CYP2E1 since it has a relatively high Km value for ethanol. In this AOP four different KEs are used, which are oxidative stress (KE1), lipid peroxidation (KE2), unfolded protein response (UPR) (KE3) and apoptosis (KE4). Oxidative stress can be defined as the imbalance between ROS and defence mechanisms against these ROS. ROS levels in a cell can rise which leads to damage by the oxidizing free radicals. Lipid peroxidation is a form of direct damage to lipids in the cell membrane or organelle membranes. The cell membrane will eventually break due to the build-up of all the damage. MDA  and 4-hydroxynonenal (HNE) are two products of lipid peroxidation. UPR is a reaction activated by stress in the endoplasmic reticulum (ER). ER stress can be induced by too much protein folding which reaches a higher level than the folding capacity. Also accumulation of unfolded protein in the ER and protein adducts formation with important endoplasmic proteins can induce ER stress, which activates UPR. The final KE is apoptosis, which is programmed cell death in general. The process of apoptosis is well regulated and several signal proteins are known to induce the apoptotic process.


Background (optional)

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Summary of the AOP

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Events: Molecular Initiating Events (MIE)

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Key Events (KE)

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Adverse Outcomes (AO)

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Sequence Type Event ID Title Short name
1 MIE 1508 CYP2E1 Activation CYP2E1 Activation
2 MIE 1509 Protein Adduct Formation Protein Adduct Formation
3 KE 1510 Oxidative Stress in Brain Oxidative Stress in Brain
4 KE 1511 Lipid Peroxidation Lipid Peroxidation
5 KE 1512 Unfolded Protein Response Unfolded Prortein Response
6 KE 1513 General Apoptosis General Apoptosis
7 AO 1514 Neurodegeneration Neurodegeneration

Relationships Between Two Key Events
(Including MIEs and AOs)

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Title Adjacency Evidence Quantitative Understanding
CYP2E1 Activation leads to Oxidative Stress in Brain adjacent Moderate Moderate
Oxidative Stress in Brain leads to Lipid Peroxidation adjacent High High
Lipid Peroxidation leads to Protein Adduct Formation adjacent High High
Protein Adduct Formation leads to Unfolded Prortein Response adjacent Moderate Moderate
Oxidative Stress in Brain leads to Unfolded Prortein Response adjacent Moderate Moderate
Lipid Peroxidation leads to General Apoptosis adjacent High High
Unfolded Prortein Response leads to General Apoptosis adjacent High High
General Apoptosis leads to Neurodegeneration adjacent High High

Network View

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Stressors

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Life Stage Applicability

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Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens NCBI

Sex Applicability

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Overall Assessment of the AOP

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Domain of Applicability

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Essentiality of the Key Events

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Key Event

Essentiality

MIEa (Protein Adduct Formation)

Moderate support. Activation of MIEa induces increased activation of KE 3, but direct evidence is not available. One theory about the mechanism is that adducts are formed with critical ER proteins. (Haberzettl, P. & Hill, B. G., 2013; Galligan, J. J. et al., 2014; Cumaoglu, A. et al., 2014; Kessova, I. G. & Cederbaum, A. I., 2005; Huličiak, M. et al., 2012; Sadrieh, N. & Thomas, P. E., 1994; Shin, N. Y. et al., 2007; Sapkota, M. & Wyatt, T. A., 2015; Tuma, D. J., 2002)

MIEb (CYP2E1 Activation)

High support. Direct evidence is available which prevents the upstream KE 1. CYP2E1 knockout as well as inhibition studies are performed. Activation of CYP2E1 by stressors also showed an increased. (Valencia-Olvera, A. C. et al., 2014; Haorah, J. et al, 2008; Luo, J., 2014; Yang, L. & Cederbaum, A., 2011; Lakshman, M. R. et al., 2013; Jimenez-Lopez, J. M. & Cederbaum, A. I., 2005; Gonzalez, F. J., 2005; Albano, E. et al., 1996; Albano, E., 2006; Wu, D. et al., 2012; Cederbaum, A. I., 2010; Lu, Y. et al., 2010; Oneta, C. M. et al., 2002; Lieber, C. S., 2004; Emerit, J. et al., 2004)

KE 1 (Oxidative Stress)

High support. Direct and indirect evidence is available for the essentiality of KE 1. Blocking ROS formation inhibits upstream KE 2 and KE 3. The indirect evidence showed that higher ROS induction showed an increased activity of upstream KE 2 and KE 3.

KE 2 (Lipid Peroxidation)

High support. There is much indirect evidence available showing that inducement of lipid peroxidation can increase activity of MIEb and KE 4. The direct evidence of blocking HNE which results in inhibition of upstream KE 4 shows that there is link between KE 2 and KE 4 and that the underlying molecular pathway is known.

KE 3 (UPR)

Moderate support. There is direct as well as indirect evidence available which shows molecular understanding of how KE 3 can induce KE 4. The uncertainty lies in whether ER stress alone can induce KE 4, or that KE 1 also plays a role in it. This is more discussed in detail in chapter 4.

KE 4 (Apoptosis)

High support. Neurodegeneration is the loss of neuron cells in the brain.

See table below where an overview is provided of the direct and indirect evidence. For the meaning of numbering see Abstract and the image of the AOP,

Key event relatio-nship

Methods

Influence on downstream Key events

Direct/Indirect evidence

KER 1: MIEb ---> KE1

 

1. Stimulation of CYP2E1 by stressors in rat livers.

2. Inhibition studies of CYP2E1 in neuron cells.

3. CYP2E1 KO in mice where TBARS values are measured.

4. Induction of CYP2E1 results in higher ROS levels an higher CYP2E1 expression, study performed in granule neuron cells.

1. Activation KE 1

2. Inhibition KE 1

3. Inhibition KE 1

4. Activation KE 1

1. Indirect

2. Direct

3. Direct

4. Indirect

KER 2: KE1 ---> KE2

 

1. Lower ROS level by adding higher concentrations of antioxidants or resveratrol (inhibitor of ROS). TBARS and LOOH product was measured in rat microsomes.

2. Correlation study where higher ROS levels increased lipid peroxidation in aging brains.

1. Inhibition KE 2

2. Activation KE 2

1. Direct

2. Indirect

KER 3: KE1 ---> KE3

 

1. Lower ROS levels by overexpression of antioxidant SOD1, NAC or GSH resulted in induction of UPR markers. Measured in neuron cells.

2. Stimulation of ROS formation by ethanol, which induces the UPR response in 2 hours after exposure.

1. Inhibition KE 3

2. Activation KE 3

1. Direct

2. Indirect

KER 4: KE2 ---> MIEa

 

1.Proteomic detection techniques for HNE adducts, HNE is a reactive aldehyde product of lipid peroxidation.

2. SERS monitoring detection, showed link between increased lipid peroxidation and increased protein adduct formation.

1. Activation MIEb

2. Activation MIEb

1. Indirect

2. Indirect

KER 5: MIEa ---> KE3

 

1. HNE (known to form protein adducts) treatment in rat aortic smooth muscle cells induced expression of the PERK pathway, which is part of the UPR. Same study is also performed in different settings.

2. Some toxicants can form protein adducts with ER proteins, what can induce ER stress and the UPR.

1. Activation KE 3

2. Activation KE 3

1. Indirect

2. Indirect

KER 6: KE2 ---> KE4

 

1. HNE can induce Fas/CD95DR expression, which regulated the extrinsic pathway of apoptosis.

2. Knockout of GSTA4 in mouse, which is an antioxidant for HNE, showed an increase in Fas expression.

3. ASK1 and JNK are activated by Fas. Increased HNE concentrations showed higher expression of ASK1 and JNK. When Fas was inhibited apoptosis was stopped.

4. HNE induces mitochondrial dysfunction which leads to apoptosis. Higher HNE levels showed increased expression of cytochrome c and caspases. Caspase 3 and 9 are mainly activated. Both are part of the intrinsic pathway of apoptosis.

1. Activation KE 4

2. Inhibition KE 4

3. Activation KE 4

4. Activation KE 4

1. Indirect

2. Direct

3. Indirect

4. Indirect

KER 7: KE3 ---> KE4

 

1. Higher expression of IRE1 and PERK, which are UPR markers, showed an increase of caspases expression. These caspases play a major role in the apoptotic pathway.

2. Inhibition of ER stress by DHCR24 resulted in a lower level of CHOP expression. Also an inhibition of apoptosis was shown.

1. Activation KE 4

2. Inhibition KE 4

1. Indirect

2. Direct

KER 8: KE4 ---> AO

1. Neuron loss is detected in neurodegenerative diseases, such as Alzheimer.

1. Activation AO

1. Indirect


Evidence Assessment

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Quantitative Understanding

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In AOP1 there are some knowledge gaps present which is one of the principles of the AOP concept. CYP2E1 activation is known to increase the ROS concentration in a cell, but the underlying mechanism is not completely understood. There are two main mechanisms which are suggested in literature, either CYP2E1 or NADPH oxidase could be the primary enzyme which is responsible for ROS formation and cause the further damage in the cells. NAPDH oxidase recycles the NADP+ which is formed during the reaction cycle of CYP2E1, during this cycle ROS is formed due to the uncoupling reaction. CYP2E1 shows a relatively high activity of NADPH oxidase activity and is poorly coupled with NADPH-cytochrome P450 reductase. When NADPH oxidase is inhibited by anti-CYP2E1 IgG a reduction of ROS induced lipid peroxidation was shown. Knock-out or inhibition of CYP2E1 itself resulted in lower oxidative stress. A study performed by Bradford et al. showed that NADPH oxidase knock-out mice attenuated liver injury, where CYP2E1 knock-out mice did not show attenuating of liver injury. On the other hand, NADHP oxidase knock-out mice did not reduce oxidative stress damage to DNA, where CYP2E1 knock-out mice did reduced the damage. Another study by Zhang et al. looked at the influence of NADPH oxidase, an inhibiter against NADPH oxidase was used which reduced the formation of ROS in PC12 cells. Finally, Shah et al. and Furukawa et al. also showed that NADPH oxidase inhibition leads to a reduced formation of ROS, both studies were done in different disease context. The principle of ROS formation by NADPH oxidase is the formation of H2O2 since O2 is used as a substrate. By the Fenton-Weiss-Haber reaction multiple oxidants can be produces. But as mentioned above, several studies showed that CYP2E1 inhibition alone is enough to reduce ROS formation. To take into account, studies described above are all done in liver cells. The mechanism of CYP2E1 activation could be different in the brain.

Another knowledge gap is the mechanism of protein adducts that can induce ER stress, and ultimately the UPR. The assumed mechanism is that protein adducts are formed with critical ER proteins, which leads to the dysfunction of the ER. Furthermore, it is also a possibility that protein adducts inhibit the folding of proteins. These proteins can accumulate in the ER and when the protein accumulation is higher than the capacity ER stress is induced. Further research must be done to define the mechanism of how ER stress is induced by protein adducts, which will eventually lead to the UPR.


Considerations for Potential Applications of the AOP (optional)

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References

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